Autoantibodies to insulin are part of the prodrome of type I or insulin dependent diabetes mellitus (IDDM) and antibodies to human insulin routinely accompany treatment of diabetes. To understand the genetic and structural features of insulin antibodies, anti-insulin B cells were isolated from a treated IDDM patient and the nucleotide sequences of expressed VH and VL genes determined. The data indicate that most anti- insulin V genes are somatically mutated and a set of related VH-III genes is preferentially expressed. Importantly, these anti-insulin VH genes are distinct from VH-III genes found in developmental or fetal repertoires that are frequently used by autoantibodies in systemic diseases. There are no data on the genetic origins or structure of insulin autoantibodies in IDDM. However, studies in the NOD mouse indicate that abnormal expression and regulation of fetal VH genes are present in IDDM. Accordingly, this project will test the hypothesis that clonal expansion and somatic mutation of developmentally regulated VH genes play a key role in loss of tolerance and generation of insulin autoantibodies in IDDM. Specifically, the aims are to characterize the structure and function of anti-insulin B cells in IDDM by first producing IgG anti-insulin clones, and using-reverse transcriptase/PCR to identify VH and VL structures in a cohort of IgG anti-insulins. Molecular probes from the IgGs will then be used to identify clonally related elements in anti-insulin cDNA libraries and in memory B cells from the donor. A second aim is to use these techniques to test the hypothesis that exogenous insulin skews the anti- insulin repertoire to express V genes different from those selected by IDDM. Genetic elements that clonally dominate insulin autoimmunity will be identified and the contribution of germline or somatically derived structures to disease-related specificities established. These new genetic and structural markers may improve diagnosis and therapy of IDDM and will provide a means to monitor the effects of aggressive insulin therapy in pre-diabetics.